This proposal addresses three examples of novel RNA-protein interactions for which the self-amplifying RNA genome of poliovirus provides a simple experimental system and a sensitive assay for events that RNA molecules experience in cytoplasmic environments. Genetic studies of RNA recombination among poliovirus genomes have provided strong support for a copy-choice mechanism, in which recombinant RNAs result from a change of templates by the viral RNA-dependent RNA polymerase during negative strand synthesis. The analysis of RNA recombination in vitro is proposed, in order to define the activities responsible for template switching and to ask if RNA replication complexes from other cells and viruses display similar activities. In addition, DNA templates will be constructed to test the idea, suggested by genetic studies in Escherichia coli and Drosophila melanogaster, that DNA-dependent RNA polymerases can also synthesize recombinant RNA molecules, given particular DNA structures as templates. A poliovirus mutant has been isolated which displays a temperature-sensitive defect in a final stage of morphogenesis. At this stage, proteolytic cleavages occur that have been suggested from analysis of the poliovirus and rhinovirus crystal structures, to be autoproteolytic and partially RNA-dependent. Physical and genetic studies of this poliovirus mutant are planned to study the proposed autoproteolysis reaction, including sequencing of a possibly altered cleavage site and analysis of second site revertants. Certain in vivo experiments with poliovirus RNAs suggest the possible existence in animal cells of a double-stranded RNA unwinding enzyme, whose synthesis may be induced by double stranded RNA. In vivo and in vitro experiments are proposed to look for such an activity, and to determine its functions in poliovirus infection and in the host cell. The proposed research has several health-related aspects. Rearrangements in viral RNA genomes, such as RNA recombination between poliovirus vaccine strains and the generation of defective interfering particles, can have dramatic effects on pathogenesis by these viruses. Studies of a poliovirus mutant conferring thermostability to the viral capsid may be helpful in vaccine development, and investigation of the response of animal cells to double-stranded RNA may contribute to our understanding of the pathways by which animals combat viral disease.